On the basis of the analysis of 64 glycosyltransferases from 14 species we propose that several successive duplications of a common ancestral gene, followed by divergent evolution, have generated the mannosyltransferases and the glucosyltransferases involved in asparagine-linked glycosylation (ALG) and phosphatidyl-inositol glycan anchor (PIG or GPI), which use lipid-related donor and acceptor substrates. Long and short conserved peptide motifs were found in all enzymes. Conserved and identical amino acid positions were found for the alpha 2/6- and the alpha 3/4-mannosyltransferases and for the alpha 2/3-glucosyltransferases, suggesting unique ancestors for these three superfamilies. The three members of the alpha 2-mannosyltransferase family (ALG9, PIG-B, and SMP3) and the two members of the alpha 3-glucosyltransferase family (ALG6 and ALG8) shared 11 and 30 identical amino acid positions, respectively, suggesting that these enzymes have also originated by duplication and divergent evolution. This model predicts a common genetic origin for ALG and PIG enzymes using dolichyl-phospho-monosaccharide (Dol-P-monosaccharide) donors, which might be related to similar spatial orientation of the hydroxyl acceptors. On the basis of the multiple sequence analysis and the prediction of transmembrane topology we propose that the endoplasmic reticulum glycosyltransferases using Dol-P-monosaccharides as donor substrate have a multispan transmembrane topology with a first large luminal conserved loop containing the long motif and a small cytosolic conserved loop containing the short motif, different from the classical type II glycosyltransferases, which are anchored in the Golgi by a single transmembrane domain.

The underlying causes of type I congenital disorders of glycosylation (CDG I) have been shown to be mutations in genes encoding proteins involved in the biosynthesis of the dolichyl-linked oligosaccharide (Glc 3 Man 9 GlcNAc 2 -PP-dolichyl) that is required for protein glycosylation. Here we describe a CDG I patient displaying gastrointestinal problems but no central nervous system deficits. Fibroblasts from this patient accumulate mainly Man 9 GlcNAc 2 -PP-dolichyl, but in the presence of castanospermine, an endoplasmic reticulum glucosidase inhibitor Glc 1 Man 9 GlcNAc 2 -PP-dolichyl predominates, suggesting inefficient addition of the second glucose residue onto lipid-linked oligosaccharide. Northern blot analysis revealed the cells from the patient to possess only 10 -20% normal amounts of mRNA encoding the enzyme, dolichyl-P-glucose:Glc 1 Man 9 GlcNAc 2 -PP-dolichyl ␣3-glucosyltransferase (hALG8p), which catalyzes this reaction. Sequencing of hALG8 genomic DNA revealed exon 4 to contain a base deletion in one allele and a base insertion in the other. Both mutations give rise to premature stop codons predicted to generate severely truncated proteins, but because the translation inhibitor emetine was shown to stabilize the hALG8 mRNA from the patient to normal levels, it is likely that both transcripts undergo nonsensemediated mRNA decay. As the cells from the patient were successfully complemented with wild type hALG8 cDNA, we conclude that these mutations are the underlying cause of this new CDG I subtype that we propose be called CDG Ih.

Type I congenital disorders of glycosylation (CDG I) are diseases presenting multisystemic lesions including central and peripheral nervous system deficits. The disease is characterized by under-glycosylated serum glycoproteins and is caused by mutations in genes encoding proteins involved in the stepwise assembly of dolichol-oligosaccharide used for protein N-glycosylation. We report that fibroblasts from a type I CDG patient, born of consanguineous parents, are deficient in their capacity to add the eighth mannose residue onto the lipid-linked oligosaccharide precursor. We have characterized cDNA corresponding to the human ortholog of the yeast gene ALG12 that encodes the dolichyl-P-Man:Man 7 GlcNAc 2 -PP-dolichyl ␣6-mannosyltransferase that is thought to accomplish this reaction, and we show that the patient is homozygous for a point mutation (T571G) that causes an amino acid substitution (F142V) in a conserved region of the protein. As the pathological phenotype of the fibroblasts of the patient was largely normalized upon transduction with the wild type gene, we demonstrate that the F142V substitution is the underlying cause of this new CDG, which we suggest be called CDG Ig. Finally, we show that the fibroblasts of the patient are capable of the direct transfer of Man 7 GlcNAc 2 from dolichol onto protein and that this N-linked structure can be glucosylated by UDP-glucose: glycoprotein glucosyltransferase in the endoplasmic reticulum.

AGS3 contains GoLoco or G-protein regulatory motifs in its COOH-terminal half that stabilize the GDP-bound conformation of the ␣-subunit of the trimeric G i3 protein. The latter is part of a signaling pathway that controls the lysosomal-autophagic catabolism in human colon cancer HT-29 cells. In the present work we show that the mRNA encoding for AGS3 is expressed in human intestinal cell lines (Caco-2 and HT-29) whatever their state of differentiation. Together with the full-length form, minute amounts of the mRNA encoding a NH 2 -terminal truncated form of AGS3, previously characterized in cardiac tissues, were also detected. Both the endogenous form of AGS3 and a tagged expressed form have a localization compatible with a role in the G␣ i3 -dependent control of autophagy. Accordingly, expressing its non-G␣ i3 -interacting NH 2 -terminal domain or its G␣ i3 -interacting COOH-terminal domain reversed the stimulatory role of AGS3 on autophagy. On the basis of biochemical and morphometric analysis, we conclude that AGS3 is involved in an early event during the autophagic pathway probably prior to the formation of the autophagosome. These data demonstrate that AGS3 is a novel partner of the G␣ i3 protein in the control of a major catabolic pathway.Macroautophagy or autophagy is a general and evolutionary conserved response to starvation and stress activated in eucaryotic cells (1)(2)(3)(4)(5). During the induction of autophagy, portions of the cytoplasm are rapidly sequestered to form an autophagosome by a membrane of unknown origin (6, 7). After receiving input from endocytic vesicles (8 -10), autophagic vacuoles ultimately fuse with the lysosomal compartment where the sequestered material is degraded.There is increasing evidence for the importance of autophagy in tissue-specific functions such as the generation of pulmonary surfactant (11), the maturation of erythrocytes (12), and the production of neuromelanin in brain (13). Dysregulation of autophagy is associated with a variety of disease including tumor progression (14), cardiomyopathy and myopathy (15-17), neurodegenerative diseases (18,19), and bacterial and viral infections (20 -22). Recent progress, including the identification of the molecular machinery and signaling pathway involved in autophagy have brought some clues on its role in proliferation, differentiation, and cell death (23,24).Previous studies from our laboratory have shown that a cytoplasmic heterotrimeric G i3 protein regulates autophagy in the human colon cancer HT-29 cell line (25). The rate of autophagy is minimal when the G␣ i3 protein is in the GTP-bound form and becomes stimulated when GDP is bound to the G␣ i3 protein (26). In agreement with these results, GAIP, 1 a RGS protein (regulator of G protein signaling) (27), that activates the hydrolysis of GTP by the G␣ i3 protein has been shown to increase the rate of autophagy (28). More recently, we have shown that an extracellular signal-regulated kinase 1/2-dependent phosphorylation of GAIP stimulates its activity toward the GTP-boun...

During protein N-glycosylation in mammalian cells, free oligosaccharides (fOS) are generated from lipid-linked oligosaccharides by a pyrophosphatase activity and oligosaccharyltransferase and from misfolded glycoproteins by peptide:N-glycanase in both the ER and cytoplasm. Trafficking machinery comprising oligosaccharide-specific ER and lysosomal transporters, an endo-beta-N-acetyl-glucosaminidase, and the cytosolic M2C1 mannosidase drives a flux of fOS from the ER to cytoplasm and from the cytoplasm into lysosomes where fOS are degraded. Transport of fOS out of the ER is normally efficient and if inhibited causes fOS to be secreted via the Golgi apparatus. By contrast, fOS clearance from the cytosol into lysosomes is less efficient resulting in low micromolar concentrations of fOS in the cytoplasm. Structural analysis of cytosolic fOS reveals oligosaccharide families whose relative abundance highlights the importance of different ER-associated degradation (ERAD) pathways for misfolded glycoproteins and suggests that in liver cells substantial amounts of glycoproteins destined for ERAD may transit early compartments of the Golgi apparatus. Glycoprotein quality control and ERAD are controlled by N-glycan/lectin interactions and the fOS trafficking pathway would seem to ensure that fOS do not interfere with these processes which occur in both the ER and cytoplasm. Although Saccharomyces cerevisiae strains harbouring mutations in genes of the yeast fOS metabolic pathway do not display obvious phenotypes, mammalian fOS are quantitatively more important and the processes leading to their regulation are more complex, raising the possibility that distinct phenotypes will be seen in mammalian cells or animals in which fOS metabolism is modified.

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Abstract. The biosynthesis of sucrase-isomaltase was compared in enterocyte-like differentiated (i.e., grown in the absence of glucose) and undifferentiated (i.e., grown in the presence of glucose) HT-29 cells. Unlike differentiated cells, in which the enzyme is easily detectable and active, undifferentiated cells display almost no enzyme activity and the protein cannot be detected by means of cell surface immunofluorescence or immunodetection in membrane-enriched fractions or cell homogenates. Pulse experiments with L-[35S]-methionine show that the enzyme is, however, synthesized in these undifferentiated cells. As compared with the corresponding molecular forms in differentiated cells, the high-mannose form of the enzyme in undifferentiated cells is similarly synthesized and has the same apparent Mr. However, its complex form is less labeled and has a lower apparent Mr. Pulse-chase experiments with L-[35S]methionine show that, although the enzyme is synthesized to the same extent in both situations, the high-mannose and complex forms are rapidly degraded in undifferentiated cells, with an apparent half-life of 6 h, in contrast to differentiated cells in which the enzyme is stable for at least 48 h. A comparison of the processing of the enzyme in both situations shows that the conversion of the highmannose to the complex form is markedly decreased in undifferentiated cells. These results indicate that the absence of sucrase-isomaltase expression in undifferentiated cells is not the consequence of an absence of biosynthesis but rather the result of both an impaired glycosylation and a rapid degradation of the enzyme.

5 patients with CDG Ik are described, and their identification reveals that in France, this disease and CDG Ib (mannose phosphate isomerase deficiency: OMIM 602579) are the most frequently diagnosed CDG I after CDG Ia (phosphomannomutase 2 deficiency: OMIM 601785) and substantiate previous observations indicating that this disease presents at the severe end of the CDG I clinical spectrum.